New thio-derivatives of colchiceine compounds and a process of making same



United States, Paten fifice 2,820,029 Patented Jan. 1 4, 1958 W um-ERIVATIVE F L CE NE com: POUNDS nun A PROCESS or MAKING SAME I GeO fg S.Muller andLeonVelluz, Paris, France, assignors to U C. L. A. F., Paris,France, a corporation of France.

No Drawing, Application February 3, 1955 Serial No. 486,036

la msr in y, arpli a i nF a ce Februa y 1 10 Claims. (Cl. 260 210) Thepresent invention relatesrto new ,colchiceine .de-

riv tives and more praticularly toneWthio-deriyatives of colchiceinecompounds and to. a process of making same.

It is one object of the present invention to providenew.

and valuable compounds of the colchiceine series and more particularlythio colchiceine compounds derivatives.

Another object of the present invention is to provide 1 m le nd v c pwes es of dus n c e n valuable thiocolchiceine compounds and theirderivatives.

A further object oi the present invention is to provide new and valuablepreparations of low toxicityto beused.

in, industry and agriculture, for instance,v 'formodityin g" terials tocultivated soil, or by treatingseeds said pure materials or in the formof preparations con-1 taining the said materials dilutedin a sol ventorcarried by a, carrier.

Other objec ts of thepresent invention and advantageous features thereofwill become apparent as description proceeds.

In principle, the, new compounds. according, to, the. present inventionare compounds of the. colchieeine series; in which the methoxy group(-OCI-l of the tropolone nucleus C ofcompounds of the following Formula1 is replaced by a substituted or unsubstituted alkyl mercapto groupS-alkyl)l The resulting new compounds corre; spondto the followingFormula II:

In-said formulas:

R is hydrogen, an alkyl radical, an acyl radical, a carbohydrateradical, or a substitutedcarbohydrate radical;

R is hydrogen, an alkyl radical, or an acyl radical; and

R is analkyl radical or a substituted allcyl radical.

'l hegeneral Formula I is written in agreement with the acceptedformulae for. colchicine (R=CI-I desrnethylcolchicine (R= H) accordingtosantavy and Reichs 'tein,

Helv. Chim. Acta. 1950, vol. 33, page l605; and cotihicoE' side (R=C H Othe production ofwhi chiis described and claimed in copending patentapplication Serial No. 332,115 by Paul Bellet and GastonAmiard, filedJanuary 19, 1953, and which has been shown to be related to desrnethyl-colchicine (R= H Following the work on nomenclaturqbyHorowitnand Ullyot (I. Am. Chem. Soc., 1952, vol. 74, page 587),

2.- it ispossible to assign Formula-II.- to the newallcylthiocolchiceine compounds of the present invention.

The new compounds are advantageously distinguished over the colchicinederivatives previously used -by'thei r markedly lower toxicity. As isevident from the following table, the therapeutic indices,'for instance,of N-desacetyl" thiocolchicine and its N-forrnyl derivative is muchhigher than those -of..colchicine and of N-des acetyl-Nkrnet'liyl'colchicine. These indices are obtained by taking the ratio of lethaldose to anti-kariokynetic dose.

TABLE Lethal Anti There.- dtt'se I kart vpeutic DL50 kynetic Index w conc1i1..'; 2 '1L1 2 N-d'csacetyl-N-methyl calchicine 16.5 3 5.5N-QESGQGtYI'IhiJCQIChiOIntL- 210. 10 21 N-desacetyl-N{Jrmylthiocelc v1(I 0 5 20 The new com ounds can The r ekiti enf by various methods,According to ,one process for,th e production oi c omp'ounds accordingto the presentinventipna com pound having .the general Formula I isreacted witha substituted or unsubstituted alkyl mercaptan, thereaction.

being etiected at or vslightly be low roo m tempetatureor at alkylmercaptan, which may. be substituted, with. a des' methyl colchicine(Formula I, R =H), followed by alkylation or introduction of acarbohydrate radical which may be acylated, it being understood thattheflgrouping -"-S. alkyl must not be affected during the course o'fthisreaction.

In accordance with another processvfor the production of-compoundsaccording to the present invention it is possible to obtain thesecompounds. by. starting from. a derivative of iso-colchiceine havingthefgeneral Formula III below,

, NRC 0.011;,

wherein R is hydrogen, an alk l-radicahandespecially the methyl radical,a phenyl mo nocarboxylicv acid radical, or a.carbohydrate radical andespecially a hexose radica l, which may be acylated, and R,, ishydrogen, an alkyl radical, and especially a lower alkyl radical or analiphatic orarornatic a'cyl radical'and especially a,

acid 7 radical.

When a substituted or unsubstituted allty l mercaptan is reacted withsuch an iso-colchiceine compound iii-the presence of an acid catalystunder conditions similar to those employed in the reaction of compoundsof the colchiceine series, the reaction does not proceed by simplereplacement of the group OR by the group --S- alkyl; instead,rearrangement takes place simultaneously and, in the final products, theketo group and the alkyl thio group take up the positions indicated inFormula II.

In this way iso-colchicine, and ethers and esters of iso= colchiceinegive rise to substituted or unsubstituted alkyl thiocolchiceines ofFormula II.

When operating in the presence of an acid catalyst, preferably ofp-toluene sulfonic acid, benzene sulfonic acid, a hydrogen halide, orboron trifiuoride, the reaction between the colchicine compound or theiso-col-.

chicine compound described above and the mercaptan may be effected inthe presence of a solvent or in the absence thereof, at a temperaturearound room temperature or somewhat higher, preferably between about C.and about 50 C. In the case of methylmercaptan the reaction is carriedout in a sealed tube when working on a small scale and in an autoclavewhen working'od' an industrial scale. Depending on the reactivity of the(Formula II, R=H,

R=H, R =CO.CH R =substituted or unsubstituted alkyl radical). Saiddesmethyl alkyl thiocolchiceine compound is readily methylated by theaction of diazomethane yielding thereby alkyl thiocolchiceine (FormulaII, R=-CH R =CO.CH R =substituted or unsubstituted alkyl radical).

When it isdesired to obtain a heteroside of an alkyl thiocolchiceine(Formula II, R=carbohydrate radical which may be acylated, R =-CO.CH R=substituted or unsubstituted alkyl radical) an alkali metal salt of asubstituted or unsubstituted alkyl mercaptan is reacted tropolonecompound and the temperature of the reaction,

contact with the mercaptan and the catalyst is maintamed over a periodof time varying between several hours and several days. When a solventis used, it is preferably selected in such a way that the startingmaterial is brought into solution.

When the reaction is complete, the reaction mixture is cooled and excessof mercaptan is removed, for instance, by distillation. the unreactedmercaptan and to use it again. solvent is used, this-is separatedaccording to known processes. The resulting thio-derivative may bepurified by crystallization; usually it is obtained with a good used,the reaction is effected under these same conditions, but the yield isgenerally lower and sometimes it is necessary to have recourse tochromatography in order to purify the reaction mixture obtained.

In this manner it is possible to recover When a with the correspondingheteroside of colchiceine or with itsacylated derivative;'alternativelyan aceto bromo carbohydrate is condensed with desmethyl alkylthiocolchiceine andthe acyl groups of the heteroside of the alkylthiocolchiceine obtained thereby are subsequently saponified. a

Compounds of Formula II in which R, is hydrogen, an alkyl-radical, oranother acyl radical than the acetyl radical and R a substituted orunsubstituted alkyl radi- "cal, are obtained by subjecting an alkylthiocolchicine The reaction may also be effected in an alkaline meeanol, or water-miscible ethers, such as tetrahydrofuran, Y

or dioxane. This process has certain advantages over the one using anacid catalyst in the case where methyl mercaptan is employed asreactant. salts of methyl mercaptan are not volatile and, therefore, itis possible to work in an open vessel. The alkali metal salts of themercaptan may be produced separately and introduced when the reaction iseffected. Itis also possible to produce extemporaneously in aqueous oralcoholic solution of the mercaptan salt by reacting the mercaptan witha titrated solution of an alkali metal hydroxide or alternatively withsodium methylate or ethylate, and to use this solution directly. Afterreaction, the reaction mixture is acidified, diluted with water, and thethio derivative formed is extracted with a solvent which is immisciblewith water. Purification is carried out by fractional crystallization orchromatography. Usually the yields are excellent and very pure compoundsare obtained when proceeding in this manner. This process The alkalimetal of Formula II wherein R is an acetyl radical and R; a substitutedor unsubstituted alkyl radical, to acid hydrolysis" or alcoholysis. Whenthe radical R is unstable under the reaction conditions (for instance,in the case 5 of acetyl propionyl, benzoyl, trityl, glucosyl orgalactosyl yield. When compounds of the iso-colchiceine series areradicals), an N-desacetyl desmethyl alkyl-thiocolchiceine is obtained(structural Formula II, R=H; R =H; R substituted or unsubstituted alkylradical). Said compound, if required, can be alkylated, acylated, orconverted into the glucoside at the phenolic oxygen atom wherebyN-desacetyl alkyl thiocolchiceines (structural Formula II, R=alkyl,acyl, acylated or unacylated carbohydrate radical; R =H; R =substitutedor unsubstituted' alkyl radical) are obtained. When, however, theradical R is stable in the presence of acid reagents, there is obtainedan N-desacetyl alkyl thiocochiceine (structural Formula II, R=alkyl; R=I-I; R =substituted or unsubstituted alkyl radical). It is alsopossible, without departing from the spirit of the present invention, tosubject the starting material first to an alkaline hydrolysis or toanyother suitable reaction which sets free the phenolic hydroxyl groupor which is capable of modifying its substitution and subsequently to anacid hydrolysis thereby desacylating the acetamino group and producingthe corresponding amine.

The N-desacetyl thiocolchiceine compound obtained in this manner isseparated from non-reacted starting material by making use of theirdifferent solubility in of working in alkaline medium is particularlyuseful In this way des The process in accordance with the presentinvention leads with excellent yields to N-desacetylated derivativeshaving the structure of thiocolchicine.

The amino group may be alkylated or acylated with another acyl groupthan the acetyl group before or after conversion into thiocolchiceine,i. e. by alkylation or acylation either directly of N-desacetylcolchiceine or subsequently by alkylation or acylation of N-desacetylthiocolchiceine.

The structure of the new N-desacetyl alkyl thiocolchiceine compounds andN-desacetyl desmethyl alkyl thiocolchiceine compounds is proven by thefact that on treating said compounds with acetic acid anhydride in thepresence of pyridine there are obtained, due to acetylation of the aminogroup, or, respectively, due to acetylation of the amino and the phenolgroup, the corresponding alkyl thiocolchiceine compounds or thecorresponding acetyl desmethyl alkyl thiocolchiceine compounds. The lastnamed compounds yield desmethyl alkyl thiocolchiceine compounds bysaponification, in an alkaline medium, of the phenolic acetoxy group. itis, of course, possible to obtain alltyl thiocolchiceine compounds fromsaid desmethyl alkyl thiocolchiceine compounds by methylation.

The following examples illustrate the present invention, without,however, limiting the same thereto. The melting points given in saidexamples were determined by slowly heating to melting the respectivecompounds. The power of rotation was determined in chloroform at aconcentration of 0.5% by weight unless otherwise stated; All percentagesare by weight.

Example 1.-Thicolchict'ne (Formula II, R=R =-CH R =-CO.CH starting fromcolchicine 2.5 g. of colchicine and 0.5 g. of p-toluene sulfonic acidmonohydrate are introduced into a scalable tube. The lower portion ofthe tube is put into a mixture of solid carbon dioxide and acetone and,when it is well cooled, 15 g. of methyl mercaptan, cooled to 30 C., areadded thereto. The tube is sealed, is removed from the refrigerationmixture, and its temperature is allowed to rise to room temperature (20C.) while agitating the tube from time to time. Colchicine dissolvesfairly easily while p-tolucne sulfonic acid dissolves only after about 2hours. The homogeneous solution which is first of yellow color changesto red without forming a precipitate. The sealed tube is allowed tostand for 6 to 12 days at 20 C., is then cooled to 80 C., is opened, andthe mercaptan is distilled off by heating to about 20 C. to about 40 C.When it is desired to effect a fresh reaction it is of advantage torecover excess mercaptan in another sealable tube cooled to 50 C. or 80C., to again introduce into this second tube the required amounts ofcolchicine and p-toluene sulfonic acid monohydrate and to again effectthe reaction as described above.

The residue from the distillation of the mercaptan and, when present, ofthe solvent is obtained in the form of a thick resin which is red incolor and very unpleasant in smell. This residue is taken up in amixture of 50 cc. of chloroform or ethyl acetate and 20 cc. of water,sodium bicarbonate is added till a neutral reaction is obtained. Theaqueous phase is separated from the solvent phase and is again extractedwith the same solvent. The combined solvent extracts are washed withwater.

After drying the extract with magnesium sulfate and distillation todryness there are obtained 2.6 g. of a waxy residue which isyellow-brown in color and which is recrystallized from ethyl acetate.The weight of the final product obtained is 2.1 g., corresponding to 80%yield of the theoretical yield. The condensation with methyl mercaptanmay also be effected in the presence of about volumes of a solvent suchas chloroform or tetrahydrofuran, and this solvent is driven off as soonas the mercaptan has been distilled off. The thiocolchicine is presentin the form of cubic crystals having a yellow color anda melting pointof 192-194 C.; [a] =221. Thiocolchicine is very soluble in chloroform,well soluble in ethanol, and soluble inacetone. It is dissolved by 15volumes of hot ethyl acetate and is insoluble in ether It is stable inan aqueous alkaline medium up to 100 C. This stability differentiatesthe new compound from colchicine. The tendency of solvation is as markedas in the last mentioned compound.

ANALYSIS C H O NS found: C% 63.7, H% 6.0, N% 3.2, S% 8.1- Calculated: C%63.6, H% 6.1, N% 3.4, S%7.7-

Example 2.Thioeolchicine (Formula II, R=R3=C.H.3, R =--CO.CH startingfrom colchicine 20 g. of sodium hydroxide in the form of pellets aredissolved in water in such a way that a total volume of cc. is obtained;25 g. of methylmercaptan are introduced into said sodium hydroxidesolution while cooling, the mixture is allowed to stand at 0 C. for fourhours, and the resulting sodium salt of methyl mercaptan is filtered offby suction. 2.5 g. of said sodium salt of methyl mercaptan are thenintroduced into a solution of 5 g. of colchicine in cc. of water. Themixture is stirred mechanically, in order to cause dissolution. Thereaction mixture is allowed to stand at 20 C. for 16 hours and themixture is then poured into water and is extracted therefrom withseveral portions of chloroform. The chloroform extracts are combined andwashed until neutral and, after drying the chlorotorm solution overmagnesium sulfate, distillation is effected until dry. The residue istaken up in benzene. Chromatography over alumina gives a chloroformeluate contaimng 2.1 g. of thiocolchicine, melting point 192 C.; La]=22l. Said compound is identical with the compound described inExample 1. There is recovered, on the other hand, 50% of the colchicineused which is retained, adsorbed to the adsorption column, more stronglythan thiocolchicine. The overall yield of the thiocolchicine in this wayreaches 80% of the theoretical yield.

Example 3.-Thiocolchicine (Formula II, R=R3=CH3, R1=CO.CH3) startingfrom colchicine Variant (a).-5 g. of colchicine are dissolved in 100 cc.of water and the resulting solution is added to a. solution obtained bythe reaction of 15 g. of methyl mercaptan with 12 g. of sodium hydroxidedissolved in 50 cc. of water. The reaction is carried out as in Example2 and thiocolchicine is obtained with the same yield. s

Variant (b).5 g. of colchicine dissolved in 100 cc. of water are mixedwith a solution obtained by reacting 15 g. of methyl mercaptan with 17g. of potassium hydroxide in 50 cc. of water. After a reaction andworking up operation similar to the one described in Example 2, thereare obtained 25% of the theoretical yield of thiocolchicine and 55% ofthe colchicine used is recovered.

Variant (c).-5 g. of colchicine are dissolved in a mixture of 50 cc. ofwater and 50 cc. of methanol or ethanol. After heating the mixture to 30C. there is introduced with vigorous agitation a solution produced bythe reaction of 15 g. of methyl mercaptan with 7.5 g. of lithiumhydroxide dissolved in 60 cc. of water. After standing for 12 hours at30 C., there are obtained, after a reaction and working up operationsimilar to the one described in Example 2, 23% of the theoreticalquantity of thiocolchicine and 60% of colchicine which has not beenreacted and may be used again for a fresh reaction.

Example 4.Thi0colchict'rte (Formula II, R=R =-CH R =C0.CH starting fromisowolchicine Following the procedure as described in Example 1, l g. ofiso-colchicine is reacted with 10 g. of methyl mercaptan in the presenceof 0.2 g. of p-toluene sulfonic acid for 5 days. Dissolution takes placefairly rapidly and the solution which at first is yellow then changes tored. After distilling off the mercaptan, then residue is extracted bymeans of 50 cc. of chloroform. The chloroform extract is evaporated todryness, the residue is dissolved in ethyl acetate, and the solution issubjected to chromatographic adsorption on neutral alumina. Eluting iseffected with 12 portions of l00 cc. each of the same solvent. The first400 cc. give 680 mg. of anon-crystallizable product which is amorphous.The following 800 cc. give,after evaporation of the solvent,370 'mg. ofa residue which,"aftefrecrystallization from 2 cc. of ethyl acetate,melts at T87 190 C. When 'mixed with thiocolchicine having a meltingpoint of-l92 -C.--th-is material does not lowerthe melting-pointthereof.The substance obtaine'd hasall-the properties of thiocolchicineenumerated above.

Eizzrri'plef5. Thicolchicine' or'rni'zla II, R=R =-'CH 'R -CO'.CHstarting from colhic''irie 'a'ctzite ln-accordance-with the.proceduredescribed \in: Example 1, -2.5 ;g. -of:' colchiceine' acetateare --reacted with 1 0g. of

methyl mercaptan x-in 'thejpresence-of 0.5 vg. "of ep-tolne'nesulfonic-acid. .Dissolutiomtakes; place after-1% hours= and after-3-days' of contact-M 20 C. .the'mercaptan is distilled off.Thechloroform extract .freedfrom :the -acidlfraction by washing withsodium hydroxidet ields -'730=mg. of a neutralfraction which is.nen-ciystalline and-which is chromatographed in cthyl acetate solutionon a'column of 100..-g.'- of neutral alumina'suspendedin"ether. Elutionis :eifected with l'l-portions of 100 co-each of ethyl acetate. Thefractions 4, 5 and'6are=combined'and yield, after-evaporation of thesolvent, 360 mg. of a crystalline .productqnelting iin-a meltingrpointtube at 192194 C. andegivingmo depression of .the -me1ting point withthiocolchieinegsproduced inaccordance with Example 1. Said productasr'obtainedwaccording to the present example has the same rotatorypower and solubility asth'e thiocolchicine -o'lit'airied 'accdrding toExample 1. g p v The aqueous sodium'hydroxidesoliition containscolchiceine which 4 results -from'sapohification -oftlfe=startingmaterial during the course ofthe reaction. -'-In this way 1.7 g. of.-coichiceine (corresponding to 1185 g. of colchiceine acetate,equivalent ito 74%- of the starting material' used are obtained.

As desmethyl 'colchicine .is not-sufficiently soluble in methylmercaptan, 'the reaction is-eifected with its=ace tate and the acetateof desmethyl thiocolchicine 'formecl thereby is then saponified.

2.6g. of desmethyl-colchicine acetate and -O.'4:g. of ptoluene sulfonicacid monohydrate are introduced into a tube capable'of being sealed, thetube'with its contents is cooled'to -80 C.,and about -l2zg. ofme'thylmercaptan are put into the tube. The tube -is-sealed and itstemperature is allowedto rise to +20 C. Dissolution takes place moreslowly than in the case ofcolchicine. The mixture is agitated. -It isfirst of :pasty consistency and is converted into a homogeneous liquidafter 12 hours.

After standing for-seven days there is formed -a red precipitate whichadheres to the wall of the tube. The reaction mixture is left to standfor 'four more days at 20 C. which procedure appreciably increases thered precipitate. After said"11'day's, excess mercaptan is recovered aspreviously described and the red residue is taken up in 200 cc. ofchloroform. The chloroform solution is first washed with sodiumbicarbonate solution in order to eliminatethe p-toluene sulfonic-acidand then with water. After drying, the solution is evaporated to drynessyielding a residue of solvated desmethyl thiocolchicine acetate weighing3.2. g. This crude solvated acetate is dissolved in 50 cc. of 95%ethanol. 15 cc. of an N aqueous solution-of sodium'hydroxideare added tosaid solution and the resulting mixture -is allowed to stand -forminutes at 20 C. The mixture is then diluted 'with 200 cc. of water andextracted with 50 cc. of 'chlorofo'rr'n in order-to eliminatenon-phenolic substances. The redalkaline layer containing thedesmethylthiocolchicine is acidified by means of hydrochloric acid until'congo'paper is turned blue. The solution is twice extracted withchloroform. The chloroform extracts are combine'd,'dried, and evaporatedto dryness. The residue WhichweighsS g.'consists of desmethylthiocolchicine which is strongly solvate'd. This compound is dissolvedin 20 cc. of hot ethyl acetate which breaks up the chloroform 'solvate.After cooling, the desmethyl thiocolchicine crystallizes out. Saidcompound is filtered off by suction, washed with ethyl acetate, againfiltered off by suction,'and dried. 'In'thisway 1.93 g. (correspondingto a yield of 79%) of a compound melting at 305 C. areobtained."Theyellow product is soluble in alcohol, acetone, "and dimethylformamide; it dissolves in approximately 200 parts of cold chloroform.It is insoluble in water, aqueous'dilute acids, ether and benzene. Itdissolves in aqueous dilute 'alkalis giving a red solution. Desmethylthiocolchicine is purified by dissolving 600 mg. thereof in 3 cc. ofdimethyl formamide at C.; there are then added 15 cc. of ethyl acetate,the mixture is filtered, and the'filtrate is allowed to slowlycrystallize. In this way 400 mg. of pure desmethyl thiocolchicinemelting at 308 C. are obtained; [a] =--Z49i2. Theseph'ysical propertiesdo not vary after repeated recrystallization.

ANALYSIS --Found: -C% 62.9, H% 5.8, N% 3.3, S% 7.5. C H O NS calculated:C% 62.8, H% 5.8, N% -3.5, S% 8.0.

Example 7.-D'esm'ethyl thiocolchicine (Formula II, -R="H, 'R -'CO.CH R-CH starting from des- '--m'e't'h'yl colc'hicine :200 -mg. of desmethylcol-chicine aremixed with -'a solution of 200 mg. of the sodium salt ofmethyl mercaptan,-obtained-in accordance with Example 1, dissolved in 4cc. of water. T he reaction mixture is shaken until a homogeneoussolution is obtained. After standing for one night the solution is runinto water, acidified 'with hydrochloric"- acid; extracted with severalportions of chloroform, washed with sodium carbonate solution and withwater, the chloroform extracts are combined, dried, and distilled todryness.

The resulting residue gives, on repeated recrystallization from ethylacetate and acetone, mg. of desmethyl thiocolchicine in the pure state,corresponding to a yield of 70%; melting point: 308 C., [a] =249.

Example 8.Desmeth;vl thiocolchicine (Formula II,

R=H, R ='CO.CH R =-CH starting from desmethyl colchicine acetate 220mg."0f desmethyl colchicine acetate are treated with the sodium salt'ofmethyl mercaptan under conditions as described in Example 7. During thecourse of the reaction saponification takes place and, after extractionwith chloroform and purification under the conditions hereinbeforedisclosed, pure desmethyl thiocolchicine is obtained. This'c'ompoundcauses no depression of the melting point when'mixed with the compoundobtained in accordance with the method described in Example 7.

Itis,'of' course, understood that the variants described in Examples3(a), (b) and (c) (used for the production of thiocolchicine) "may alsobe used for the production of desmethyl thiocolchicine.

Example '9.-Thi0c0lchicine' (Formula II, R=R =-CH R =-CO.CH startingfrom desmethyl thiocolchicine 1.6- gJof'desmethyl thiocolchicine,produced according to 'Example '6,-"ar'e'dissolved lI1'40 cc. ofmethanol and 30 cc. -'of an 0.6% "solution of diazomethane in'tolueneare added thereto. *Therea'ction is fairly vigorous-at the beginning andthe temperature is kept within the range of 10 to 20 C. by applyingexter'nal cooling. When the reaction has calmed down, an excess ofdiazomethan e-is added and the reaction mixture is then lefttost-andfor9 two more hours. To the orange-colored solution result ing therebythere are added 2 cc. of acetic acid in order to destroy the excess ofdiazomethane. The methylation proceeds quantitatively as washing of thetoluene solution by means of sodium hydroxide does not extract anyphenolic substance. The toluene solution is then washed with water, isdried, and is distilled to dryness in a vacuum. In this way 1,640 g. ofcrude thiocolchicine are obtained; this material, on recrystallizationfrom ethyl acetate to which an equal volume of ether had been added,melts at 188190 C. and does not depress the melting point ofthiocolchicine produced according to Example 1.

Example 10.Tetraacetate of thiocolchicosia'e (Formula II,R=tetraacetylglucsyl, R CO.CH R =CH starting from colchicosidetetraacetate As colchicoside is insoluble in methylmercaptan itstetraacetyl derivative (produced according to French Patent No.1,053,179) which dissolves easily therein is used as starting material.The reaction is carried out in a similar manner as described in Examples1 and 6, but using 3 g. of tetraacetate of colchicoside and 25 g. ofmethyl mercaptan in the presence of 0.4 g. of p-toluene sulfonic acidmonohydrate; the reaction mixture is allowed to stand for 5 days duringwhich time a precipitate appears. After distilling off excess methylmercaptan the residue is taken up in 100 cc. of ethyl acetate orchloroform, the solution is washed with sodium bicarbonate solution,then with water, is dried over sodium sulfate, and the solvent isdistilled ofi. There results a residue having a resinous appearance andweighing 2.82 g. which does not crystallize from the usual solvents butwhich is sufficiently pure for hydrolysis to desmethyl thiocolchicine orfor saponification to thiocolchicoside.

Example 11 .Thi0colchic0side (Formula 11, R=C H O R --CO.CH R ==CHstarting from thiocolchicoside tetraacetate Y 2.7 g. of thiocolchicosidetetraacetate are dissolved in 42 cc. of ethanol and 7 cc. of N sodiumhydroxide solution are added. The resulting reaction mixture is allowedto stand for 24 hours at 20 C., is then neutralized and extracted with amixture of chloroform and ethanol (8:2). The resulting extract is washedwith water, dried, and evaporated to dryness. The then resulting residueconsists of thiocolchicoside. After triturating this compound with 10volumes of ether, it is filtered by suction and yields, after drying,1.2 g. of thiocolchicoside in the form of a powder which crystallizefrom ethyl acetate. F=215 C; [a] ==550i1O (0.5% concentration in waterExample 12.Desmethyl thiocolchicine (Formula II,

R=H, R =CO.CH R =-CH starting from thiocolchicoside tetraacetate 30 cc.of methanol are used to dissolve 1.5 g. of crude thiocolchicosidetetraacetate (obtained according to Example 10) and 120 cc. of 0.2 N ofhydrochloric acid are added. The methanol is evaporated and the reactionmixture is heated on a water bath for 2 hours during the course of whichthere is formed a black precipitate. The mixture is cooled, sodiumhydroxide solution is added until the pH value is above 10, and theresulting alkaline solution is extracted with chloroform in order tofree it from non-phenolic substances. The sodium hydroxide solution,which has a red color, is acidified with hydrochloric acid and isextracted with chloroform. After drying and evaporation to dryness thereremains a residue of 480 mg. of .desmethyl thiocolchicine in the crudestate, corresponding to a yield of 58% of theory. On recrystallizationfrom 2 to 3 cc. of dimethyl formamide to which cc. ofethyl acetate hadbeen added, there result 420 mg. of desmethyl thiocolchicine melting 'at305 C. A mixture of said compound with the highly P re product obtainedin accordance with Example 6 (melting point 308 C.) melts at 306 C.which proves that the two substances are identical.

Instead of starting with thiocolchicoside tetraacetate it is alsopossible 0 hydrolyze according to the method of the present example thethiocolchicoside obtained in accordance with Example 11; this also givesrise to desmethyl thiocolchicine.

Example I3.-Thiocolchicoside (Formula II, R=C H O R =CO.CH R =-CHstarting from colchicoside 400 mg. of colchicoside are introduced into asolution of 400 mg. of sodium methyl mercaptide in 2 cc. of water whilevigorously agitating. After allowing the mixture to stand for 16 hoursat 23 C., the orange solution is acidified with acetic acid and thismixture is extracted several times with a mixture of chloroform andethanol (1:1). The extracts are combined, dried over potassiumcarbonate, and then distilled to dryness. The thiocolchicoside in thecrude state obtained in this way is purified by recrystallization firstfrom ethanol and then from ethyl acetate. The product is obtained with ayield of 50% and is present in the form of small squares or octagonalprisms having a clear yellow color and melting at 215 C., [a] =-550i10(0.5% concentration in Water). The thiocolchicoside, identical with theproduct described in Example 11, is soluble in 1 volume of water oraqueous acidsor alkalies. It is also soluble in 20 volumes of ethanolwith which the product gives a solvate containing 1 mol of ethanol. Theproduct is insoluble in ether, acetone and benzene.

ANALYSIS Found: C% 57.6, H% 6.1, 0% 28.5, N% 2.3, 8% 5.7. C H O NS(=563.6) requires C% 57.5, H% 5.9, 0% 28.4, N% 2.5, S% 5.7. It is, ofcourse, understood that this compound may also be produced according tothe variants d6SCl'lb6d'fQ1' Example 14.Thi0c0lchicoside (Formula. II,R: C5H11O5, R1=-CO-CH3, R =-CH starting from desmethyl thiocolchicine500 mg. of desmethyl thiocolchicine (Formula II, R=H, R CH producedaccording to the method of Example 7 are dissolved in 2.5 cc. of Nsodium hydroxide solution. The resulting solution is cooled to 0 C. and700 mg. of crystallized tetraacetyl bromo glucose dissolved in 3.5 cc.of acetone are added thereto. The resulting red solution is allowed tostand for 15 hours and 25 cc. of weakly alkaline water is added thereto.Chloroform is used to extract the resulting mixture, washing is effectedwith water, and drying over magnesium sulfate. The colchicosidetetraacetate obtained in this way is directly saponified by 0.03 cc. ofalcoholic potassium hy-- starting from colchicine 2 g. of colchicine aredissolved in 5 cc. of chloroform and a suspension of 300 mg. ofp-toluene sulfonic acid in 4 cc. of ethyl mercaptan' (boiling. point 37C.) are added to the resulting solution. The reaction mixture is stirreduntil dissolutiontakes placefandds 'all'owe'd- 'to stand in'aclosedreceptacle for! several days at 20 C. Evaporation to dryness is effectedand'the residue is taken up in chloroform, 'the resultingsolution beingwashed, dried and evaporated to dryness. The residue is crystallizedfrom ethyl acetate and yields 12 g. (54%) of'e'thyl thiocolchiceinehavinga -melting point of 207 208 C., [a] 226. -The'comPorind isobtained-in the form of yellow crystals which are insoluble inwater, ether andpetroleum ether, but soluble in chloroform, methanol, ethanol, acetone,and dimethylformamide.

ANALYSIS Example I6.Production of ethylthioeolchiceine (Formula III,R=CH R =CO.CH R =-C H starting from colchicine 2 g. of colchicineare-dissolved in 4 cc. ofethyl mercaptan, 300 mg. of benzene-sulfonicacid are added, and the resultingmixtureis allowed to standinaclosed'receptacle for one week at-20 C. The resulting precipitate isfiltered with suction'and is crystallized from a mixture of acetone andwater. Ethyl thiocolchiceine-is Obtainedthereby. -The compound 'isidentical with-the compound described inExample- 15. The-yield-is 35%.

Example 17.Productin of ethyl thiocolchiceine (Formula II, R=CH3,R1=-CO.CH3 R2=-CZH5) starting from colchicine 2 g. of colchicine aredissolved in 10 cc. of tetrahydrofuran, first 4 cc. of ethylmercaptanand then afew drops of concentrated hydrochloric acid are added, and theresulting mixture is allowed to stand in a closed receptacle for 6 daysat room temperature. After treatment as indicated in Example 15 ethylthiocolchiceine is obtained with a yield of 25%.

Example 18.Pr0 aacti0n of ethyl thiocolchiceine (Formula R:CH3,R1='-CO-CH3, R =C H starting from colchicine .mercaptan inBO cc; of Nsodium hydroxide solution are added'and. the resulting mixture-is'al-lowed 'to stand for 2 days atroom temperature. The reaction mixtureis extracted withchl'oroform. -After' 'wo'rkingupthe extract asdescribed: inExample 15; ethylthiocolchiceine is obtained in'ayield of35%.

Example"20.-Pr0dacti0n' of ethyl thiocolchiceine (Formula R=CH3,R2=-C2H5) starting from colchicine 2 g. of colchicine are dissolved'in40 cc. of water. A solution of 1.5 g. of ethyl mercaptan in 30 cc. of Npotassium hydroxide solution is added and the resulting mixture isallowedmo stand for 2 daysf'at' room temperature. The rea'etion mixtureis extracted with chloroform. The chloroform extract is worked up 'asd'e's'crib'edin Example 15 whereby ethyl thiocolchiceine is. obtained ina yield Example ZIP-Production of ethyl thiocolchiceine (Foring fromcolchicine "2- g. of colchicine are dissolved in 40 cc. of water. 'Asolution of'1.5- g. of ethyl'mercaptan in 30 cc. of N lithiumhydroxidesolution is added and the resulting mixture is allowedto standfor 2 daysat room temperature. -The reaction mixture is'extracted withchloroform and the extract is worked up as described in Example 15.Thereby ethyl'thiocolchiceine'is obtained with a yield of 30%.

Example 22.2-hydr0xy ethyl thiocolchiceine (Formula II, R= --CH R=-CO.CH R =CH -CH OH) starting from colchicine 8 g. of colchicine aredissolved in 16 cc. of dry chloroform. First 16 cc. of mercapto ethanoland-then 1.2'. g. of p-toluene sulfonic -acid are added to saidsolution. After standing for seven days at room temperature, the mixtureis poured into water, extracted with chloroform, and the resultingsolution is dried and evaporated to dryness. The resulting residue istaken up in'80 cc. of boiling ethyl acetate and this solution is allowedto crystallize. 5.1 g. (55%) of 2-hydroxy ethyl thiocolchiceine of themelting. point 225-228 C; are obtained. The product is recrystallizedfrom a mixture of ethanol and ether- (1:3). The compound is insoluble inwater, ether, petroleum ether, but is soluble in ethanol, methanol,chloroform, and dimethylformamide. Melting point 234 C.; [a] =214.

ANALYSIS Found: c% 61.9, H% 6.3, N% 3.1, s% 7.5. C23H27OGNS requires C%62.0, H% 6.11, N% 3.14, 5% 7.2

Example 23. 2-hydroxy ethyl thiocolchiceine (Formula II, R=-CH R =CO.CHR =-CH -CH OH) starting from colchicine the one obtained in theprecedingexample.

Example 24.-N- desac etyl thi ocaIc/zicine (Formula I,

R="-CH R =H,-"R =CH )-'starting from thiocolchicine To 4.88 g. ofthiocolchicine there are added 6O cc. of methanol and 60- cc.! of 2" Nhydrochloric ac'id. The mixture is heated under reflux for 18' hours.Thereafter 40 cc. of. aqueousmethanol .are 'distilled offand theremaining solution. is extractedthree times using cc. of chloroform eachtime. .The chloroform- -extracts are combined-andwashedrfourtimes using100 cc; of water each time. Thewashed chloroform extract is dried anddistilled-to dryness,".-yie1ding 400 -mg.- ofcrude thiocolchicine.

The aqueous layer, which is extracted with chloroform, and the washwaters are combined, mixed with 10 N sodium hydroxide solution untila'pH'value of 13 is achieved, andthen extractedfivetimes each with 100 cc.of chloroform. The combined chloroform extractsare washed twice with 50cc. of Water'each time, dried, and

distilled to dryness. 1 A residue weighing 5.2 g., due to the presenceof-chloroform of 'solvation, is obtained. This residue isataken-up in-20 cc. of chloroform and-" cc. of ether are added theretogcrystallization sets in immediately yielding 3 .52 g. of-desacetylthiocolchicine {F01 agaaiomaa form) are heated under refiux for-24hoursin 60 cm. of

methanol and 60 CCuOf hydroohloric a'cid (2 N). After distilling-off themethanol the mixture is. neutralized with sodium bicarbonate and: isextracted with chloroform. The. chloroform extract. is evaporated tordryness. Recrystallization of the residue from CflJBDOlLYlldS'3J1"g-(80% yield) of the pure compound having a melting point of 185 C., [a]'=2l3 (0.5% concentration in *ethanol) The N-desa'cetyl" desmethyhthiocolchicine is' obtain'ed inrthe form of prismatic needles of ayelloworange color. It is soluble in 15*partsby volume of hotethanol-and l'pa-rts by volume of cold/ethanol, in chloroform, but isinsoluble in ether,- petroleum ether, and water. It is soluble inalkalies andacids due'toits-ampho teric nature which 'derivesfrom thepresence of "ana'mirio group and a phenol group, both in -thetfree=.state,..in-its molecule.

ANALYSIS form and, after purification according toExample 25, N'-

desa'cetyldesmethyl thiocolchicine is obtainedwitha yield of 60%. Thephysical and chemical properties of this compound are .the same rasthoseof the compound-adescribed in Example 25.

Example 27.Producti0n'-.:"o;f:N=desacetyl desmethyl thiocolchicinestarting from thiocolchicoside. "tetraacetate by meansof fractionalhydrolysis The procedure is the same as set forth in Example 26, but thehydrolysis is interrupted after heating for-six hours. The resultingdesmethyl'thiocolchicine" i's -"then extracted with chloroform. Afterwashin'g, drying; and evaporation, there is obtained desmethylthiocolchicine having a melting point of 308- C. [a] =249,'with a yieldof 60%.

The chloroform wash waters are combined'with the aqueous hydrolysislayer. a The mixture is neutralized'with sodium bicarbonate. extractedwith chloroform, and the compound obtained from said chloroform extractsis purified as set forth in Example: .25; 1 In this wa'y, N- desacetyldesmethyl thiocolchicine with a 1' yield of J 12% is obtained drolysisof thiocolchieasidelatraacetate 21f gsiof .thioedlehieoside:tetnaacetate.are'saponified' by means :35 ice. otrethanolnand 6 fccn'o f N sodiumhydroxide solution for about 24 hours at room temperatures Thereactiontmixture.iszthenvacidifiedtbymeansof 2--Nhydrochlorictaoid-zand'is'heatedunderirefltixfor 36 hours.Subsequentatreatment is reflected irl the samemanner as set forth inExample 25. The yield ofsNedesacetyl desmethyl thiocolehicine isvExample 29:=1 rodubtibn*-0f N-d'esacetyl-klesmethyl 'Ihiorolcliicinestarting from thiocdl'chz'coside 2 ..g. otthiocolchicoside arehydrolyzed ,bymeans of liydrochloric'acid during 140. hours Underftheconditidns set forth inExample 26. jTheTliydroehlorie, acid. solution,after extraction with chloroform; yieldsf79'% oif lN-desacetyl desmethylthideolchiciheWhich islidenticalwith the compound obtained according toExample. 25..

I. Into. a. scalable tube: tthere-zaretintroduced 13.2%.; of N-desaeetyl: thiocolchicine rand-.1 gs-ofnbenzaldehyde. A reactionoccurstimmediately:betweensthe:amino groupof N-desaeetylthio'colchieine: and-,;:b.enzaldehyde,-:: thus, :giv' ingthe--.corresponding .Sohilf: base; which; :iS; amorphous. The reaction:product is Ariturated with rzether; and Z1116 solvent is-.then.-tdrivenoff. :Thcn 1.16. :gm of .methylxiodide are introduced. Thetubeistsealedsand heated atii lOOf. C. for S hours.

After;;cooling, ,theireactiormproduot is'rtaken' up in -t1'00 CUQ? ohmethanol,." 100 cc. .1 of N5 sodiumrhydroxide'solution are added, andthe mixturerisrheated:at.. 50 C. :for five minutestandvextractedl with?chloroform-x: Y "The chloroform extract is dried-over. sodi'umasulfateandxthei solvent is distilled off. The residue. obtained thereby iscrude N- desaeetyl-Nmethyl-thioco1chicine, which is present in, the formlof a microcrystalliney yellow powder; melting at about 1-70-- C.-saidcompoundean' be purified 'by' chromatography over alumina-soas-to-yield--the-pure product ofthe melting-"pointi222' C.; identic'al'to-gthat described in Example 40.

Itis understood that methylation can also beefi?eeted with othermethylation reagents; for'instanceywith methyl bromide 0ft .dimethyl.sulfate. ,F-inally; itaisgpossiblecto. produce the same compound.byreactingmethyl mercaptan with N-desacety1-N-methyl.colchicine;

evaporated .to. dryness. .The residue obtained in, this .way

is dissolved in 40 cc. of ethyl acetate from which solution itcrystallizes. In this way 1.7 g. of N-desacetyl- N-formylthiocolchicine-are obtained, having amelting point of1259- C.,-:[a]'.=275, (0.'5-%=concentrat ion;

in chloroform). The product is recrystallized;forianalysis purposes,them 180 parts by volumeof e thyl acetate which is thenconc'entrated' toparts by volume, 'fol lowed by recrystallization from 20 parts by volumeof methanol, and finally from 100 parts by volume of ether.

ANALYSIS Found: C% 62.9, H% 6.0, N% 3.2, S% 8.0, 19.9. C H I NS (401.46)requires C% 62.8, I-I% 5.8, N% 3.5, S% 8.0, 0% 19.9.

The N-desacetyl-N-formyl thiocolchicine has never before been described;it is insoluble in water and ether, and is soluble in methanol, benzene,chloroform, and sparingly soluble in ethyl acetate.

Example 32.-N-desacetyl-N-f0rmyl desmethyl thiocolchicine (Formula I,R=H; R =--CH0; R =--CH starting from N-desacezyl desmethylthiocolchicine 6 cc. of pur formic acid and 2.4 cc of acetic acid an-Thydride are mixed with each other at a temperature of 0 C. and theresulting mixture is allowed to stand for two hours at room temperature.This mixture is slowly added to a solution of 400 mg. of N-desacetyldesmethyl thiocolchicine in 8 cc. of pyridine cooled to l0 C. Afterstanding for two hours at room temperature, the reaction mixture isdiluted with water, acidified with 2 N hydrochloric acid until a pHvalue of 2 is obtained, and extracted with chloroform. The chloroformextracts are washed with water and then with N sodium hydroxidesolution. Thereby the phenolic hydroxyl group is set free, and theresulting sodium salt passes into the aqueous phase. This aqueous phaseis again acidified, as indicated above, and extracted with chloroform.The chloroform extracts are washed with water, dried with magnesiumsulfate, and evaporated to dryness. The residue is taken up in 4 cc. ofethyl acetate from which it crystallizes. In this way there are obtained370 mg. (83%) of N- desacetyl-N-formyl desmethyl thiocolchicine, havinga melting point of 284-287 C., [a] =--268 (0.5% concentration inchloroform).

The product is recrystallized for analysis purposes from a mixture ofmethanol and ether (1:4).

ANALYSIS Found: 0% 62.3, H% 5.7, N% 3.3, S% 8.4, 0% 20.5. C H 0 NS(387.44) requires C% 62.0, H% 5.5, iN% 3.6, 8% 8.3, 0% 20.6.

The N-desacetyl-N-formyl desmethyl thiocolchicine has not been describedheretofore; it is soluble in dilute alkalies and methanol, sparinglysoluble in chloroform, and insoluble in water and ether.

Example 33.N-desacetyl-N-benz0yl thiocolchicine (Formula I, R1=COC6H5;R2=CH3) starting from N-desacetyl thiocolchicine 600 mg. of N-desacetylthiocolchicine are dissolved in 6 cc. of pyridine, the solution iscooled to 0 C. and 3 cc. of benzoyl chloride are added. The resultingmixture is allowed to stand for two hours at room temperature, isdiluted with water, extracted with chloroform, and the chloroformextracts are washed first with 2 N hydrochloric acid, then with water,thereafter with sodium bicarbonate, and again with water, and are driedover magnesium sulfate. The dried extract is evaporated to drymess. Theresulting residue is taken up in 25 cc. of ethyl acetate from which itcrystallizes. In this way 610 mg. (80%) of N-desacetyl-N-benzoylthiocolchicine, having a melting point of 283-285 C., [u] =86 (0.5%concentration in chloroform) are obtained.

The product is recrystallized for purposes of analysis from an aqueoussolution of dimethyl formamide.

ANALYSIS Found: C% 67.7, 1H% 5.7, N% 2.7, S% 6.8. C I-I O NS (477.56)requires 0% 67.9, H% 5.7, N% 2.9, S% 6.7.

The N-desacetyl-N-benzoyl thiocolchicine has not been describedheretofore; it is soluble in chloroform and Idi- 16 methyl formamide, issparingly soluble in ethyl acetate and is insoluble in water and ether.

Example 34.-N-desacetyl-N.O-dibenz0yl desmethyl thiocolchicine (FormulaI, R=COC l-I R =COC H R =CH starting from N-desacetyl desmethylthiocolchicine 800 mg. of N-desacetyl desmethyl thiocolchicine aredissolved in 24 cc. of pyridine, the resulting solution is cooled to 0C., and 8 cc. of benzoyl chloride are added thereto. After allowing themixture to stand for two hours at room temperature, cc. of water areadded, the mixture is extracted with chloroform, and the chloroformextracts are washed first with 2 N hydrochloric acid, then with water,thereafter with N sodium hydroxide solution, again with water, and arefinally dried over magnesium sulfate. The dried extract is evaporated todryness. The residue is taken up in 20 cc. of ether, to which 100 cc. ofpetroleum ether are added. In this way, 900 mg. (72%) ofN-desacetyl-N.O-dibenzoyl desmethyl thiocolchicine in crystalline form,having a melting point of 264-266 C., [a] =-114 (0.5% concentration inchloroform) are obtained. For analysis purposes, the product isrecrystallized from an aqueous solution of dimethyl formamide.

ANALYSIS Found: C% 70.0, H% 5.2, N% 2.6, 5% 5.8, 0% 17.4. C H 0 NS(567.63) requires C% 69.8, H% 5.2, N% 2.5, S% 5.7, 0% 16.9.

The N-desacetyl-N.0-dibenzoyl desmethyl thiocolchicine has not yet beendescribed; it is insoluble in water and ether, sparingly soluble inacetone, and soluble in chloroform and dimethyl formamide.

Example 35.N-desacetyl-N-benz0yl desmethyl thiocolchicine (Formula I,R=H; R =COC H R =CH starting from N-desacetyl-N.O-dibenz0yldesmethylthiocolchicine.

500 mg. of N-desacetyl-N.0-dibenzoyl desmethyl thiocolchicine, obtainedaccording to Example 34, are dissolved in 15 cc. of ethanol, and 4 cc.of N sodium hydroxide solution are added. The resulting mixture isallowed to stand for two hours at room temperature, 50 cc. of Water areadded, and the mixture is extracted twice with chloroform. The aqueoussolution is acidified with 2 N hydrochloric acid to a pH-value of 2,extracted with chloroform, the chloroform extracts are washed with wateruntil the last traces of acid have disappeared, dried over magnesiumsulfate, and evaporated to dryness. The residue is taken up in 5 cc. ofethyl acteate from which it crystallizes. In this way, 200 mg. ofN-desacetyl-N- benzoyl desmethyl thiocolchicine, having a melting pointof 252254 C., [a] =38 (0.5% concentration in chloroform) are obtained.

The product is recrystallized for analysis purposes from acetone.

ANALYSIS Found: 0% 67.2, 11% 5.6, N% 2.8, 8% 7.0, 0% 17.7. C H 0 NS(463.53) requires C% 67.4, H% 5.4, N% 3.0, S% 6.9, 0% 17.3.

The N-desacetyl-N-benzoyl desmethyl thiocolchicine has not previouslybeen described; it is soluble in acetone and chloroform, but isinsoluble in Water and ether.

Ex mple 36.N-de.s1acetyl-N-carbethoxy thiocolchicine v (Formula I,R1=CO2C2H5; R2=CH3) starting from N-desacetyl thiocolchicine 1 g. ofN-desacetyl thiocolchicine is dissolved in 10 cc. of chloroform, theresulting solution is cooled to 0 C., and Sec. of ethyl chloroformate,and thereafter 10 cc. of triethylamine, are added. The mixture is heatedunder reflux during aperiod of one hour and is then allowed to stand fortwelvehours at room temperature, The re- 17 action mixture isthen-evaporated to dryness and triturated with 50 cc. of water, filteredwith suction, the residue is washed with water, dried, andrecrystallized from ether. 870 mg. (73%) of N-desacetyl-N-carbethoxythiocolchicine, havinga melting point of 195 C. and

(0.5% concentration in chloroform), are obtained.

ANALYSIS Found: C% 61.9, H% 6.2, N% 2.9, S% 6.9. C H O NS (445.52)requires C% 62.0, H% 6.1, N% 3.14, S% 7.19.

The N-desacetyl-N-carbethoxy thiocolchicine has not been describedpreviously; it is soluble in ether, acetone, benzene and chloroform, butinsoluble in water.

Example 37 .Conversion of N-desacetyl desmethyl thiocolchicine intodesmethyl thiocolchicine 425 mg. of N-desacetyl desmethyl thiocolchicineare dissolved in a mixture of 20 cc. of pyridine and 20 cc. of aceticacid anhydride. After standing at room temperature, dilution with water,and extraction with chloroform, the chloroform extract is washed, dried,and distilled to dryness. The resulting residue is then treated with 1.2cc. of N sodium hydroxide solution and 7 cc. of ethanol. After dilutionwith water, carbon dioxide is bubbled through the solution in order toconvert the excess of sodium hydroxide into sodium bicarbonate. Theresulting solution is extracted with chloroform and the extract isevaporated to dryness. The residue is taken up in as little hot dimethylformamide as possible and ethyl acetate is added thereto. In this waythere are obtained 250 mg. of desmethyl thiocolchicine, having a meltingpoint of 308 C. and [a] =249 (0.5 concentration in chloroform), which isidentical to the compound used in Example 25 as starting material.

Example 38.-Prducti0n 0f N-desacetyl ethyl thiocolchiceine (Formula I,R=CH R =H; R =-C H 700 mg. of ethyl thiocolchiceine, obtained inaccordance with any one of Examples to 21, are dissolved in 10 cc. ofmethanol, and 10 cc. of 2 N hydrochloric acid are added thereto. Thismixture is heated under reflux for sixteen hours, is then allowed tocool, and is poured into water. The resulting aqueous solution is washedwith chloroform, made alkaline with sodium hydroxide, and extracted withchloroform. The extract is washed, dried, and evaporated to dryness. Theresulting residue is crysltallized from a mixture of ethyl acetate andether (2:3), and yields 520 mg. (83%) of N-desacetyl ethylthiocolchiceine which, after recrystallization, melts at 163 C., [a]=219 (0.5% concentration in chloroform). This compound, which has neverbeen described before, is present in the form of yellow crystals, whichare insoluble in water, ether, and petroleum ether, and are soluble inaqueous acids, chloroform, dimethyl formamide, acetone, and in 9 partsby volume of hot alcohol.

ANALYSIS Found: C% 65.3, H% 6.5, 0% 16.5, N% 3.4, S% 8.2. C H O NS(387.48) requires C% 65.1, H% 6.5, 0% 16.5, N% 3.6, 8% 8.3.

Example 39.Pr0ducti0n of N-desacetyl ethyl thiocolchiceine (Formula I,R=CH R =H; R =C H 700 mg. of ethyl thiocolchiceine are dissolved in 10cc. of methanol, and 10 cc. of 2 N hydrobromic acid are added thereto.The resulting mixture is heated under reflux for sixteen hours and isthen worked up as set forth in Example 38. In this way N-desacetyl ethylthiocolchiceine, having a melting point of 163 C., [a]

.-219 (0.5% concentration in chloroform), is obtained with a crude yield'o'f'60%. This compound'is identical with the one described in thepreceding example.

Example 40.Production of N-desacetyl-N-methyl thiocolchicine (Formula I,R=-CH R =CH 2= 3) 1.5 g. of colchicum substance F or demecolcine(isolated from meadow saffron according to the procedure disclosed by F.Santavy, Pharm. Acta Helv., 1950, vol. 25, page 248), having a meltingpoint of 183 C. (on the Maquenne bloc), [a] =127 (1% concentration inchloroform), are dissolved in 7.5 cc. of methanol. 7.5 cc. of water and1.5 g. of sodium mercaptide in the crystalline form are added thereto,and the resulting clear solution is allowed to stand for twenty fourhours at 20 C. Abundant crystallization occurs. After filtering withsuction, washing with water, and drying, the resulting product weighs1.25 g. corresponding to a yield of melting point 215 C.

By recrystallization from a mixture of methanol and water (1:1) oracetone and water, the melting point of the thiodemecolcine in the purestate reaches 222 C. (on the Maquenne bloc). [a] =164 (0.5%concentration in chloroform). The compound is present in the form ofirregular, yellow prisms, which are readily soluble in dilute aqueoushydrochloric acid, chloroform and dimethyl formamide. The substance issoluble in ethanol and methanol, is sparingly soluble in ethyl acetate,and is insoluble in water, ether, and petroleum ether.

ANALYSIS FOIJlIld: (3% 65.3, H% 6.7, N% 3.4, 5% 8.0. C21H2504NS requires65-1, 6.5, 3.6, 8% 8.3.

Example 41 .Production of N-desacetyl-N-methyl thin colchicine (FormulaI, =-CH R =-CH 2= 3) 1.5 g. of naturally occurring colchicum substance Fare dissolved in 7.5 cc. of water and 8 cc. of an aqueous solutioncontaining 1.2 g. of potassium hydroxide and 1.1 g. of methyl mercaptan,which solution was previously prepared at 0 C., are added thereto. Afterallowing the mixture to stand for twenty four hours at 20 C., it isdiluted with water, extracted with chloroform, and washed with water.After drying the solution and evaporating the solvent in a vacuum, theresidue weighing 1.5 g. is present in the form of an amorphous powderhaving a melting point of approximately C., which, after taking up inmethanol and dilution with water (1:1), gives, on seeding with a sampleproduced according to Example 40, N-desacetyl-N-methyl thiocolchicine orthiodemecolcine, having a melting point of 222 C., with a yield of 50%.The compound is identical in every respect with the one obtained in thepreceding example.

In place of methyl and ethyl mercaptan used in the preceding examples,there can be employed equimolec- -ular amounts thereof primary andsecondary alkyl mercaptans such as n-propyl-mercaptan, isopropylmercaptan, n-butyl mercaptan, isobutyl mercaptan, amyl mercaptan,mercapto propanol, mercapto butanol. The reaction of these mercaptanswith colchiceine and isocolchiceine compounds is otherwise carried outin the same manner as described in said preceding examples.

In place of the acetate of colchiceine as used in Example 5 and theacetate of desmethyl colchiceine as used in Examples 6 and 8 there canbe used equimolecular amounts thereof of an acylated colchiceine anddesmethyl colchiceine compound corresponding to the above given FormulaI but having another acyl group than the acetyl group, such as thepropionates, the butyrates, the

benzoates, the carbomethoxy, the carbethoxy derivatives of colchiceineand desmethyl colchiceine compounds while otherwise the procedure" isthesame as set forth in said examples.

In place of formic acid as used in Examples 31 and 32, of benzoylchloride as used in Examples 33 and 34, or of chloro formic acid ethylester as used in Example 36, there can be employed equimolecular amountsthereof of other 'acylating agents such as propionic acid, propionylchloride, butyroyl chloride, toluic acid chloride, chloro formic acidmethyl ester, and others. Otherwise, the procedure during such acylationis the same as set forth in said examples. V

The new thio-derivatives of colchiceine compounds according to thepresent invention are used, for instance, in agriculture in the form oftheir solutions in suitable solvents or in mixture with solid carriermaterials. Concentrations between 0.02% and 0.2% have proved to be orgreat value in the treatment of seeds to produce polyploidism.

Saidnewthio derivatives of colchiceine compounds accordiij'g to thepresent'invention as they are disclosed herein, are also "of therapeuticuse in human as' well as in veterinary'medicineL I In human medicinethey are topicallyapplied, 'for' instance, in 'the' treatment of inflammatoryor allergic diseases of the skn such as pruritus anderythematous edematous conditions, and are also useful for modifying ofthe ka'ryokinesis of the bone marrow and for the treatment of othermitotic'disorders. Inveterinary medicine the new compounds have provedof value in the topical treatment of inflammatory diseases of the skin,such as dog s 'eczema and for advantageously modifying the chromosomesin breeding'animals resulting in increased vitality and acceleration ofcross-breeding. It may be mentioned that ordinarily colchiceinecompounds can be reacted with alkyl mercaptans'i'nthe presence of acidcatalysts as well as with an alkali metal salt of saidalkyl mercaptanwhile isocolchiceine compounds always require the presence of an acidcatalyst" and do not react satisfactorily with the alkali metal salt ofsaid alkyl mercaptans.

The term acyl as used hereinbefore and in the claims annexed heretoindicates not'only the acyl radicals derived, for instance, from loweraliphatic acids and phenyl monocarboxylic acids, but also'carbalkoxy'radicals derived from carbonic acid. I 7

Of course, many other changes and variations in the starting materials,the reaction components, the reaction conditions, temperature, andduration, themode of working up the reaction products and of isolatingand purifying the same may be made by those skilled in the art .inaccordance with the principles set forth herein and in the claimsannexed hereto.

'We claim:

1. As a new compound, a thiocolchiceine compound of the formula li r e hhiqs teisaia at th t me HaGO- HaCO NEE-GOSH:

S.CH2.OHzOH 3. Thiocolchicoside of the formula S .O Hs 4. N-desaeetylthiocolchicine of the formula SCHs 7;. A process according to claim 10,wherein the reaction with the lower alkyl mercaptan is carried out at atemperature between about lO" C. and about 50 C.

8. A process accordingto claim 7, wherein the reaction with the loweralkyl mercaptan is carried out in the presence of an acid catalyst.

9. A process according to claim 7, wherein the reaction of a colchiceinederivate with the lower alkyl men captan is carried out with an alkali'metal salt of the alkyl mercapt'an.

10. In a process of producing a thiocolchiceine compound of the formula21 wherein:

R is a member selected from the group consisting of hydrogen, the methylradical, the benzoyl group, the glucosyl group, and the tetraacetylglucosyl group,

R is a member selected from the group consisting of hydrogen, the methylradical, a lower alkanoyl group, the benzoyl group, and the carbethoxygroup, and

R is a member selected from the group consisting of a lower alkylradical and a hydroxy (lower) alkyl radical,

the steps comprising heating in a sealed tube an excessof a lower alkylmercaptan with a compound selected from the group consisting of acolchiceine compound of the formula CH:.O NH-CO.CHB

CHM

CH: and an isocolchiceine compound of the formula CH|.0 NH.OO.CH!

in which formulas:

R represents the same member as indicated above,

R is a member selected from the group consisting of hydrogen, the methylradical, the benzoyl group, the glucosyl group, and the tetraacetylglycosyl group, and

R is a member selected from the group consisting of hydrogen, the methylradical, a lower alkanoyl group, the benzoyl group, and the carbethoxygroup,

until formation of the thiocolchiceine compound of the above givenformula is completed, and isolating the reaction product from thereaction mixture.

References Cited in the file of this patent Santavy et al.: Helv. Chim.Acta, vol. 33, 1950, pages 1611, 1614 to 1627.

Goldberg et al.: Cancer, vol. 3 (1950), pages 124 to 129.

Lettre Angewandte Chemie, vol. 63 (1951), pages 422 to 424.

1. AS A NEW COMPOUND, THIOCOLCHICEINE COMPOUND OF THE FORMULA
 3. THIOCOLCHICOSIDE OF THE FORMULA 